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目的探讨生物钟PER1基因在口腔鳞癌中的昼夜节律变化情况和与肿瘤体内生长的关系。方法 60只裸鼠置于12 h光照和12 h黑暗交替环境中饲养3周后,将人颊鳞癌BcaCD885细胞接种于裸鼠颊部,建立口腔颊鳞癌模型。3周成瘤后,在24 h内按灯亮后4、10、16、22 h(4 HALO、10 HALO、16 HALO、22 HALO)的4个时间点分别处死15只裸鼠,取出肿瘤,称量,常规切片在HE染色下计算各时间点肿瘤的有丝分裂指数(MI);分别用S-P免疫组化、Western blot和Real-time RT-PCR检测各时间点癌细胞中PER1蛋白和mRNA的表达;分别用方差分析和余弦分析检验各指标在4个时间点的差异性和是否具有昼夜节律性。结果颊鳞癌细胞PER1蛋白、PER1 mRNA、肿瘤MI和肿瘤质量在昼夜不同时间点具有显著性差异(P<0.01),其变化波动具有昼夜节律性特征(P<0.05);肿瘤MI和肿瘤质量与PER1的表达水平呈反比关系,PER1 mRNA表达的峰值与肿瘤MI和质量的谷值均位于活动相的中期,而PER1 mRNA表达的谷值与肿瘤MI和质量的峰值均位于休息相中期。结论口腔鳞癌中PER1的表达、肿瘤MI和质量在昼夜不同时间点的波动具有24 h昼夜节律性规律,PER1在口腔鳞癌中为抑癌基因。
Objective To investigate the circadian rhythm of PER1 gene in oral squamous cell carcinoma and its relationship with tumor growth in vivo. Methods Sixty nude mice were housed in 12 h light and 12 h dark environment for 3 weeks. Then, BcaCD885 cells of human squamous cell carcinoma were inoculated into the buccal area of nude mice to establish buccal squamous cell carcinoma model. After 3 weeks of tumor formation, 15 nude mice were sacrificed at 4 time points of 4, 10, 16, and 22 h (4 HALO, 10 HALO, 16 HALO, and 22 HALO) The expression of PER1 protein and mRNA in cancer cells at various time points were detected by SP immunohistochemistry, Western blot and Real-time RT-PCR respectively. The variance analysis and cosine analysis were used to test the differences of each index at four time points and whether it has circadian rhythms. Results PER1 mRNA, PER1 mRNA, tumor MI and tumor mass in buccal squamous cell carcinoma were significantly different at different time points between day and night (P <0.01), and the fluctuation of PER1 protein was characterized by circadian rhythms (P <0.05). The tumor MI and tumor mass The expression of PER1 was inversely proportional to the expression level of PER1. The peak of PER1 mRNA expression and the peak value of tumor MI and mass were located in the middle of the active phase, while the trough of PER1 mRNA and the peak of tumor MI and mass were located in the mid-rest phase. Conclusions The expression of PER1 in oral squamous cell carcinoma and the fluctuation of tumor mass and MI in different time points of day and night have circadian rhythm of 24 h and PER1 is the tumor suppressor gene in oral squamous cell carcinoma.